A novel superfusion device and optical imaging system to study border zone physiology in cultured heart cell monolayers

A novel superfusion device and optical imaging system are presented for the study of intracellular ion regulation and impulse propagation in monolayers of cultured cardiomyocytes at border zones. The method uses a custom dual-flow superfusion chamber to produce a gradient in metabolic inhibition (MI) over cultured cardiomyocyte monolayers to simulate the depletion of cellular ATP and the development of intracellular acidosis occurring at ischemic boundaries in the regionally ischemic heart. An ∼250 mum wide gradient in metabolic inhibition, or "border zone", was formed between the normal and MI zones. Intracellular Ca2+ and pH, mitochondrial membrane potential, ATP and cell viability were examined at the border zone. The multiparametric study provided both spatial and temporal information on the intracellular state of the cultured cardiomyocytes in the border zone region in relation to the external gradient in metabolic inhibition. The study serves as the foundation for future studies using this model to explore the impact of intracellular parameters on electrophysiological characteristics at the border zone. A multisite optical imaging system with high temporal resolution was also developed for measuring both Ca2+i dynamics and impulse propagation in this model using Ca2+-sensitive fluorescent dyes (CaSDs) and voltage-sensitive fluorescent dyes (VSDs), respectively. The optical imaging system uses silicon photodiode sensors and two optical fiber arrays. The optical fibers transmit the fluorescence signal from discrete sites on the cultured monolayer of cardiomyocytes to individual photodiode sensors. Similar optical imaging systems have frequently been used with VSDs, but have only infrequently been used to measure CaSDs. This optical imaging system affords the ability to measure not only impulse propagation but also Ca2+ i dynamics at multiple sites in cultured cell monolayers. This device will be used with the superfusion system described above to explore both Ca 2+i dynamics and impulse propagation across border zones in two-dimensional networks of cultured cardiac myocytes. In conclusion, the superfusion system and the high temporal resolution optical imaging system will facilitate the study of intercellular communication and interactions across boundaries of cardiac tissue where different ionic or metabolic conditions are present, for example, between ischemic and non-ischemic myocardium.